Tumors arise from uninhibited cell division, which includes mitosis and cytokinesis. Mechanistic study of mitosis has led to successful cancer therapies. Failure in cytokinesis leads to polyploidy and genetic instability that is often associated with tumors. Thus, mechanistic study of cytokinesis may open new avenues for designing strategies for cancer diagnosis and/or treatment. Cytokinesis in animal and fungal cells involves actomyosin ring (AMR) contraction and targeted membrane deposition. With the development of genetic model systems and genome-wide screens, a large number of components of the AMR and membrane trafficking have been identified, most of which are conserved from yeast to humans. The central challenge for the field now is to determine how these components are assembled together to form the contractile and membrane-trafficking machines that operate with high efficiency and fidelity, and how these machines are coordinated in time and space. It is within this context, we propose to address two major questions in cytokinesis using the budding yeast model. In the first Aim, we will investigate the mechanism and function of higher-order assembly of type-II myosin during cytokinesis, a fundamental question that remains poorly understood in any system. Recently, we have demonstrated by rotary-shadowing EM for the first time that Myo1, the sole type-II myosin in budding yeast, forms a two-headed structure with a kink in the middle of its tail, bearing all the major features of non-muscle type-II myosins in animal cells. Our preliminary studies also suggest that the targeting of Myo1 to the division site and the assembly of Myo1 into higher-order structures are controlled through distinct domains in its tail, in contrast to other systems where targeting and assembly of type-II myosins are coupled. Thus, the budding yeast system provides a unique opportunity to address the specific in vivo function of myosin higher-order assembly during cytokinesis. In the second Aim, we will attempt to define a novel mechanism underlying the coordination between AMR contraction and the exocytosis-mediated ECM remodeling during cytokinesis. Specifically, we will test our hypothesis that the C2-domain protein Inn1 coordinates AMR contraction and ECM remodeling or primary-septum (PS) formation in yeast by interacting with IQGAP on the AMR side and with a putative transglutaminase/protease Cyk3 on the ECM side, which, in turn, stimulates the activity of the chitin synthase Chs2 to promote PS formation at the division site. Our proposed studies will generate original insights into the role of myosin higher-order assembly in cytokinesis as well as the mechanisms underlying the coordination between AMR contraction and ECM remodeling during cytokinesis. PUBLIC HEALTH RELEVANCE: Cytokinesis is a fundamental process essential for proliferation, differentiation, and development. Failure in cytokinesis is associated with serious human diseases such as cancer. Thus, studying the mechanisms of cytokinesis will have profound implications in basic as well as clinical sciences.